An improved method for detection of apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain.
We describe a novel procedure for combining a fluorescent variant of the TUNEL technique with Hoechst 33342 stain (bis-benzimide) to identify apoptosis in tissue sections and cell culture. The biochemical hallmark of apoptosis is internucleosomal DNA cleavage, which gives rise to oligonucleosome-sized fragments (multiples of approximately 180 bp) that can be directly visualised by labelling with biotinylated or digoxygenin-conjugated nucleosides in a reaction that employs terminal deoxynucleotide transferase (TUNEL). TUNEL and Hoechst 33342 have been used separately to identify apoptosis. TUNEL specifically labels dying cells, yet a low background makes comparison of labelled cells with surrounding normal cells difficult and causes disorientation in tissue sections. Hoechst 33342 binds all DNA therefore staining all nuclear material, allowing identification of apoptotic nuclei, but the analysis is laborious. Combining the two fluorescent labels allows the initial recognition of apoptotic cells using the TUNEL technique then, by simply changing the filter, the TUNEL positive nuclei can be compared to surrounding normal nuclei to assess changes in morphology and size. Hoechst 33342 acts as a counterstain, allowing identification of anatomical structures, and permits quantitative comparison between TUNEL positive versus normal cells. We have evaluated the technique using sections of rat embryo, post-axotomy neonatal dorsal root ganglia and adult dorsal root ganglia cell culture.
Whiteside
G
G
University of Cambridge, Department of Anaesthesia, Addenbrookes Hospital, UK. gtw20@medschl.cam.ac.uk
Cougnon
N
N
Hunt
S P
SP
Munglani
R
R
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001562:Benzimidazoles; D002478:Cells, Cultured; D005260:Female; D005456:Fluorescent Dyes; D011247:Pregnancy; D051381:Rats; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology*; D016707:Tissue Fixation
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride